1. Field of the Invention
The invention is related to the detection of lipopolysaccharide-polypeptide macromolecules or portions thereof in solution and more particularly is related to methods of detecting endotoxins of gram negative organisms in fluids including biological liquids.
2. Brief Description of the Prior Art
Gram-negative septic shock is a possibly fatal development in mammals who suffer burns, undergo surgery and/or contract fulminating infections. Present theory is that at least four basic physiologic defects are associated with localized or disseminated gram-negative sepsis in humans, which differentiate the conditions from conditions of similar symptomology. These defects are an infectious process, hypotension, hypoxia and intravascular coagulation.
The condition of septic shock can develop very rapidly, often in a matter of hours to a point of irreversibility. Septic shock has poor prognosis, the survival rate being something less than 30 to 40%. When the condition of septic shock progresses to the point where a circulatory collapse is manifested by symptoms of such collapse, only about 25% of the victims survive.
For the above reasons, it is of vital importance that diagnostic tools be made available which will reliably and rapidly confirm the presence or absence of endotoxins in solutions, particularly biological fluids such as blood serum, spinal fluid, urine, saliva, pus and like materials. Time is of the essence if appropriate treatment for septic shock is to be undertaken. Accordingly, any method which provides an advantage of speed, even minutes, is of great potential value provided the method has a high degree of accuracy.
It is also of great commercial importance to make available rapid, economic, simple and reliable test procedures for the detection of endotoxins as indicative of the presence of gram-negative microorganisms in foodstuffs, pharmaceuticals, industrial fermentations and like materials.
Prior to my invention, it was known that the lysate extracted from the amebocytes of Limulus polyphemus (the horseshoe crab) coagulated in the presence of endotoxins. This knowledge was the basis of a number of methods disclosed for the detection of endotoxins. For example, in the Journal Laboratory of Clinical Medicine, 78 (1), pages 136-148, (July, 1971), Cooper et al discloses the detection of endotoxin in fluids by first admixing the test fluid with Limulus amebocyte lysate. The mixture is incubated at 37.degree. C. for from 4 to 6 hours and then at room temperature for up to 20 hours. The mixture of incubate is observed every 15 minutes during the initial 4 to 6 hour incubation period and thereafter periodically for a total of 24 hours. Development of an increase in turbidity or viscosity of the mixture is a positive sign of endotoxin presence. Similarly, Hochstein et al., Bulletin of the Parenteral Drug Association, 27 (3): 139, discloses the detection of endotoxins by admixing a test fluid with Limulus amebocyte lysate and incubating the mixture for 60 minutes at 37.degree. C. At the end of this period, gelation of the mixture is said to be a positive indication of the presence of endotoxin.
Reinhold et al. reported in Proc. Soc. Exp. Biol. and Med., 137: 334, (1971) an improved method of detecting endotoxins in blood plasma. The improved method comprises first treating the unknown plasma with acid to obtain a pH of 4.0 and then adjusting with buffer to a pH of 6.2. The unknown specimen is then diluted with normal saline. To the treated plasma there is then added a proportion of Limulus amebocyte lysate and the resulting mixture is incubated for 60 minutes at 37.degree. C. Gelation of the mixture during this period is said to be indicative of endotoxin presence.
Additional teachings of the art, generally cumulative to the above, may be found for example in Levin et al., Thrombos Diathes. Haemorrh., 19 186, (1968); Levin et al., Bulletin of John Hopkins Hospital, 115: 265, (1964); Jorgensen et al., Pro. Soc. Exp. Biol. and Med., 146: 1024-31, (1974); Wildfeuer, App. Microbiol., 28: (5): 867-71, (November, 1974); Levin et al., J. Lab. Clin. Med., 75 (6): 903,-911; Eibert, Bulletin of the Parenteral Drug Association, 26 (5): 253-260; Jorgensen et al., Applied Microbiology, 26 (1): 38-42; and Nachum et al., New England Journal of Medicine, 289 (18) 931-934, (Nov. 1, 1973).
In general, the prior art methods have required long incubation times and/or given results which are difficult to interpret, i.e.; depend upon the observation of a degree of viscosity or turbidity for a positive indication of the presence of endotoxins. A difficulty of interpretation, does not lend accuracy and reliability to the methods of the prior art. The prior art methods also leave an open question as to the exact end point of the test procedures, i.e.; a question as to the proper length of time for incubation beyond which any coagulation of the test material may be discounted as due to causes other than the presence of endotoxins.
By the method of my invention, rapid results may be obtained with clear indications of positive or negative presences of endotoxin. The method of my invention is simple to carry out, does not require extensive training, may be performed with a minimum of laboratory apparatus, gives a high degree of reproducible and reliably accurate results and is highly sensitive to even minimal quantities of endotoxins.